PROJECT SUMMARY This proposal directly responds to RFA-CA-17-017 PQ5: How does mitochondrial heterogeneity influence tumorigenesis or progression? Tumor initiation, growth, death and metastasis are associated with significant changes in cell metabolism and signaling, central to which are mitochondria. Mitochondria are complex organelles that regulate energy metabolism, signaling and apoptosis, and contain mitochondrial DNA (mtDNA) that hardwires respiration and ATP production from within. It is often underappreciated that mitochondria in different cell types, and even within individual cells of the same type, vary in function and dynamics (location, shape and movement) basally and in response to stress. It is presently unclear how these aspects of mitochondrial heterogeneity contribute to tumorigenesis. Striking changes in glucose metabolism and mitochondrial respiration occur in tumors (the ?Warburg effect?); however, these metabolic adaptations are neither uniform nor static. For example, mitochondria and metabolism of tumor cells, infiltrating immune cells and stromal cells, are diverse and responsive to ever-changing environmental stresses, including alterations in nutrient and oxygen availability, pH, and growth factors. The overarching theme of this proposal is that the heterogeneity in mitochondrial respiration, network dynamics and mtDNA-interferon (IFN) signaling not only affects cancer cell metabolism and growth, but also impacts their sensitivity to immune responses and immunotherapy. In Aim 1, unique mouse melanoma cell lines will be employed that exhibit significant differences in immunogenicity as well as mitochondrial respiration and mtDNA levels. In these cells, mitochondrial respiration will be activated or inhibited via knock-out of the Mcj or Cox10 genes, respectively, and tumor growth, immunoreactivity, and responses to anti-PD1 checkpoint blockade (immunotherapy) will be addressed to directly examine how mitochondrial heterogeneity links with immunogenicity/immunoevasion. Heterogeneity in mitochondrial dynamics and metabolism will also be assessed directly using microfluidic, single-cell approaches. Finally, as a potential therapeutic avenue to enhance anti-tumor immunity, T cells will be engineered to increase mitochondrial respiration and ATP production. In Aim 2, the focus will be on the role of mtDNA-stress mediated IFN signaling and whether it underlies differences in tumor growth, immune responses and sensitivity to immunotherapy. In Aim 3, models that pair patient-derived xenografts with their tumor infiltrating lymphocytes (PDX-TIL models) will be analyzed to probe the relevance of heterogeneity in mitochondrial respiration and mtDNA-stress signaling in human cancer. This work has the potential to illuminate new candidate pathways in tumor and immune cells to enhance anti-cancer therapies and stimulate anti-tumor immunity.